The 3D structure of rhodopsin is becoming quite clear due to input from various studies. A photoaffinity labeled analog with a fixed cis-11,12- double bond has shown that C-4 of the chromophore is near the middle of helix F. This photo-crosslinking will be repeated with other intermediates along the transduction pathway is near the middle of helix F. This photo- crosslinking will be repeated with other intermediates along the transduction pathway using a similar photolabeled analog without the fixed 11,12-double bond. The first intermediate will be bathorhodopsin which requires photo-activation to be performed at -196 degrees Celsius and the analysis of rhodopsin intermediates in subsequent stages will not be sample; however, this procedure will lead to general methods facilitating photo-crosslinking studies. In connection with these 3D investigations, we will determine the absolute sense of twist around the chromophoric C-6-s and C-12-s bonds by incorporation of enantiomeric retinal with predetermined conformations. Preliminary studies will also be continued to investigate the origin of the little understood but well-documented phenomenon of bleaching adaptation. The related dark-activity of all- trans-retinal/opsin mixtures by time-resolved difference CD and biochemical studies will be addressed. Age-related macular degeneration (AMD) is a major cause of blindness of which no remedy exists; ca. 1.7 million Americans have impaired vision from AMD. This disease involves the damage or breakdown of the macula, a small area of the retina responsible for sharp vision. With age, fluorescent chromophores accumulate in granules of retinal pigment epithelial (RPE) cells. It is possible that these pigments absorb photons in the visible range leading to light-induced damage. Two fluorescent compounds present in the granules have been characterized; they are named A2E and iso-A2E because they consist of two molecules of vitamin A aldehyde (retinal) and one molecule of ethanolamine. These compounds are present in 70 and 80 year old eyes, but not in fetal eyes. The detergent- like wedge-shaped structure of these molecules make them strong candidates for involvement in AMD. The biosynthesis and phototoxicity of A2E and RPE cells, the preparation of antibodies against A2E, and the effect of A2E on RPE function will be investigated. The anti-AMD principle(s) claimed to be present in bilberry is also under study. After defining the role of A2E in AMD, a treatment may involve the elimination of cellular A2E as well as the prevention of its formation.